Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding

Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans

ABSTRACTThere are few treatments that slow neurodegeneration in Alzheimer’s disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood–brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aβ) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aβ40 and Aβ plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4–18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial

Characterization of a re‐engineered, mesothelin‐targeted Pseudomonas exotoxin fusion protein for lung cancer therapy

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin‐targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non‐specific toxicity. To overcome both obstacles we developed RG7787, a de‐immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B‐cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin‐treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI‐H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2–3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm3) underwent remission during three treatment cycles with RG7787. Also in two patient‐derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti‐tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti‐tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors

Target binding and thermal stability.

<p>(A) Surface plasmon resonance sensogram of the Fab and CrossFab interacting with their target, hAng2. One out of three runs for the Fab and the CrossFab is shown. Coloured curves represent the measured data at various Fab or CrossFab concentrations, while the result of the global fit to a 1∶1 Langmuir model is illustrated by black curves. The table shows average values and standard deviations derived from triplicate measurements (k_a: association rate; k_d: dissociation rate; K_D: affinity). (B) Measurement of protein stability by temperature-dependent protein autofluorescence emission maximum wavelength.</p

Data collection and refinement statistics.

*<p>Values in parentheses are for the highest-resolution shell. , where is the scaled observed intensity of the <i>j</i>th observation of reflection <i>h</i>, and is the mean value of corresponding symmetry-related reflections.</p

Elbow angles and relative domain orientation.

<p>(A) Superposition of Fab and CrossFab with example structures covering the range of observed elbow angles. Example structures (shown as loops), their orientations and color coding are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Stanfield1" target="_blank">[24]</a>. All structures are superimposed via their V_L domains. The colors of Fab and CrossFab are analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a>. (B) Superposition of the CrossFab with its closest structural homolog (PDB code 3FMG). (C) Relative domain orientation of variable and constant domains in Fab and CrossFab. The pseudo-twofold axes of variable and constant domains are shown as light and dark purple dumbbells, respectively. Molecules are oriented so that the axes connecting the last Fv residues' C-alpha atoms, as well as the Fv pseudo-twofold axis are parallel to the paper plane. An asterisk marks the V_L-C_H1 junction, which is two amino acids longer than the corresponding V_L-C_L junction.</p

CrossMab design.

<p>(A) Schematic representation of the domain crossover leading to CrossFabs. Right: Combination of a Fab and a CrossFab to obtain a CrossMab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Schaefer1" target="_blank">[16]</a>. Antibody domains are symbolized as ovals. Light colors are used for LC domains; darker colors are used for HC domains. This color code is used throughout. (B) Sequence alignment of the elbow and adjacent regions of LC and HC in Fab and CrossFab.</p